Methods for identifying herbicidal compounds

ABSTRACT

The invention concerns novel enzymes having an arogenate dehydrogenase activity, in particular arogenate dehydrogenase enzymes of plants, and the genes encoding said enzymes. The inventive arogenate dehydrogenase enzymes catalyze the last stage of the metabolic pathway of tyrosine biosynthesis, and constitute, as such, potential targets of herbicides. Hence the invention also concerns a method for identifying herbicide compounds targeting said enzymes, said herbicide compounds preventing tyrosine biosynthesis by being fixed on said enzymes. The invention further concerns transgenic plants tolerant to herbicide compounds targeting an enzyme involved in the tyrosine biosynthesis pathway, in particular an enzyme involved in the transformation of L-tyrosine prephenate, in particular an arogenate dehydrogenase enzyme. Said plants become tolerant by expression in their tissues of a prephenate dehydrogenase enzyme, said enzyme being insensitive to said herbicide compounds and enabling the plant to synthetize tyrosine despite being treated with said herbicide compounds.

RELATED APPLICATIONS

This application is a national stage application (under 35 U.S.C. 371) of PCT/FROl/03832 filed Dec. 5, 2001, which claims the benefit of French application 00/15,723 filed Dec. 5, 2000.

The contents of the following submission on compact discs are incorporated herein by reference in its entirety: two copies of the Sequence Listing (REPLACEMENT COPY 1 and COPY 2) and a computer readable form copy of the Sequence Listing (REPLACEMENT CRF COPY), all on compact disc, each containing: file name: 5500-120 Sequence Listing; date recorded: Oct. 16, 2006; size 93 KB.

The present invention relates to novel enzymes having arogenate dehydrogenase activity, in particular plant arogenate dehydrogenase enzymes, and also to the genes encoding these enzymes. The arogenate hydrogenase enzymes according to the invention catalyze the final step in the metabolic pathway of tyrosine biosynthesis and, in this respect, constitute potential targets for herbicides. The present invention therefore also relates to a method for identifying herbicidal compounds having these enzymes as a target, said herbicidal compounds preventing tyrosine biosynthesis by attaching to said enzymes. The invention also relates to transgenic plants tolerant to herbicidal compounds having as a target an enzyme involved in the biosynthetic pathway for tyrosine, in particular an enzyme involved in the conversion of prephenate to L-tyrosine, in particular an arogenate dehydrogenase enzyme. These plants become tolerant by expression, in their tissues, of a prephenate dehydrogenase enzyme, this enzyme being insensitive to said herbicidal compounds and enabling the plant to synthesize tyrosine despite treatment with said herbicidal compounds.

The biosynthetic pathway for aromatic amino acids constitutes a metabolic pathway which is essential for plants, bacteria and fungi. In addition to the biosynthesis of tyrosine, phenylalanine and tryptophan, this metabolic pathway plays an essential role in the production of many secondary aromatic metabolites involved in processes such as plant-microbe interactions, the biosynthesis of structural biopolymers such as lignin and suberin, hormone synthesis, or quinone synthesis. Among all the living organisms which have this metabolic pathway, two pathways have been identified for converting prephenate to tyrosine (FIG. 1; Stenmark et al., 1974). In most chlorophyll-containing bacteria, some microorganisms and most plants, L-tyrosine is synthesized via the arogenate pathway (Abou-Zeid et al., 1995; Byng et al., 1981; Connely and Conn 1986; Frazel and Jensen 1979; Gaines et al., 1982; Hall et al., 1982; Keller et al., 1985; Mayer et al., 1985). In this pathway, the prephenate is transaminated to arogenate by a specific transaminase, prephenate aminotransferase (EC 2.6.1.57), and the arogenate is then converted to L-tyrosine by an arogenate dehydrogenase (EC 1.3.1.43; ADH on FIG. 1). In a different manner, in organisms such as the bacterium Escherichia coli or yeast, the prephenate is, initially, converted to p-hydroxyphenylpyruvate by a prephenate dehydrogenase (EC 1.3.1.12, EC 1.3.1.13), which p-hydroxyphenylpyruvate is transaminated to L-tyrosine (Lingens et al., 1967). By virtue of its role in the biosynthetic pathway for tyrosine in plants, the arogenate dehydrogenase enzyme constitutes a potential target for novel herbicides.

Other enzymes involved in this metabolic pathway already constitute major herbicide targets. Mention may, for example, be made of the enzyme 5-enolpyruvyl-shikimate 3-phosphate synthase (EPSPS), involved upstream of prephenate synthesis, which is the target for the total herbicide glyphosate. Mention may also be made of the enzyme p-hydroxyphenylpyruvate dioxygenase (HPPD) involved in the conversion of p-hydroxyphenylpyruvate to homogentisate. HPPD is the target for novel families of herbicides, the activity of which leads to bleaching of the leaves (Schulz et al., 1993; Secor 1994). These herbicides are in particular isoxazoles (EP 418 175, EP 470 856, EP 487 352, EP 527 036, EP 560 482, EP 682 659, U.S. Pat. No. 5,424,276), in particular isoxaflutole, a maize-selective herbicide, diketonitriles (EP 496 630, EP 496 631) in particular 2-cyano-3-cyclopropyl-1-(2-SO₂CH₃-4-CF₃ phenyl)propane-1,3-dione and 2-cyano-3-cyclopropyl-1-(2-SO₂CH₃-4,2,3-Cl₂ phenyl)propane-1,3-dione, triketones (EP 625 505, EP 625 508, U.S. Pat. No. 5,506,195), in particular sulcotrione or mesotrione, or else pyrazolinates.

One of the advantages of the herbicides having for a target enzymes involved in the metabolic pathways essential to plants is their broad spectrum of activity on plants of distant phylogenetic origins. However, such herbicides also have the major drawback, when they are applied to crops in order to eliminate the undesirable plants or “weeds”, of also acting on the cultivated plants. This drawback can be overcome by using cultivated plants tolerant to said herbicides. Such plants are generally obtained by genetic engineering, by introducing into their genome a gene encoding an enzyme for resistance to said herbicide, in such a way that they overexpress said enzyme in their tissues. To date, three main strategies using genetic engineering have been employed to make plants tolerant to herbicides. The first consists in detoxifying the herbicide by transforming the plant with a gene encoding a detoxification enzyme. This enzyme converts the herbicide, or its active metabolite, to nontoxic degradation products, such as, for example, the enzymes for tolerance to bromoxynil or to basta (EP 242 236, EP 337 899). The second strategy consists in transforming the plant with a gene encoding the target enzyme mutated in such a way that it is less sensitive to the herbicide, or its active metabolite, such as, for example, the enzymes for tolerance to glyphosate (EP 293 356, Padgette S. R. & al., J. Biol. Chem., 266, 33, 1991). The third strategy consists in over-expressing the sensitive target enzyme so as to produce, in the plant, large amounts of target enzyme, if possible much greater than the amount of herbicide entering the plant. This strategy, which has been used to successfully obtain plants tolerant to HPPD inhibitors (WO 96/38567), makes it possible to maintain a sufficient level of functional enzyme despite the presence of its inhibitor.

The fact that two biosynthetic pathways for L-tyrosine exist in different taxonomic groups, and in particular that the pathway directly converting prephenate to p-hydroxyphenylpyruvate is not found in plants, makes it possible to envision a fourth strategy for making plants tolerant to herbicides. Specifically, in the case of use of a herbicidal compound having as target the arogenate dehydrogenase enzyme in plants, transforming the plants intended to be made tolerant with a gene encoding a bacterial or yeast prephenate dehydrogenase enzyme will enable said plants to synthesize L-tyrosine, and therefore to tolerate the presence of the herbicidal compound despite the inhibition of the arogenate dehydrogenase enzyme by said herbicidal compound. This novel strategy therefore consists in creating, in the plants intended to be made resistant, a bypassing of the natural metabolic pathway for tyrosine biosynthesis, which pathway uses the arogenate dehydrogenase enzyme, by artificial implantation in these plants of a novel metabolic pathway for tyrosine biosynthesis, which uses the prephenate dehydrogenase enzyme. Such bypassing allows the plants possessing it, preferably plants of agronomic interest, to tolerate the presence of the herbicidal compound which inhibits the natural metabolic pathway, whereas the plants not possessing this bypassing, in particular the weeds, will be sensitive to said herbicidal compound.

DESCRIPTION

The present invention therefore relates to novel isolated polynucleotides encoding an enzyme having arogenate dehydrogenase activity. According to the present invention, the term “polynucleotide” is intended to mean a natural or artificial nucleotide sequence which may be of the DNA or RNA type, preferably of the DNA type, in particular double-stranded. The expression “enzymes having arogenate dehydrogenase activity” is intended to mean the enzymes capable of converting arogenate to L-tyrosine. The arogenate dehydrogenase activity is measured by any method which makes it possible either to measure a decrease in the amount of the arogenate substrate, or to measure an accumulation of a product derived from the enzyme reaction, namely L-tyrosine or the cofactor NADPH. In particular, the arogenate dehydrogenase activity can be measured by the method described in example 4.

According to a particular embodiment of the invention, the polynucleotides encoding an arogenate dehydrogenase enzyme comprise polynucleotides encoding the polypeptide sequence selected from the sequence described in the sequence identifier SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13. It is well known to those skilled in the art that this definition includes all the polynucleotides which, although comprising nucleotide sequences which are different as a result of the degeneracy of the genetic code, encode the same amino acid sequence, which sequence is represented by the sequence identifiers SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13.

The present invention also comprises isolated polynucleotides encoding arogenate dehydrogenase enzymes and capable of hybridizing selectively to one of the polynucleotides described above, or a fragment of these polynucleotides constituting a probe. According to the invention, the expression “polynucleotide capable of hybridizing selectively” is intended to mean the polynucleotides which, by one of the usual methods of the state of the art (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Nolan C. ed., New York: Cold Spring Harbor Laboratory Press), hybridize with the polynucleotides above, or with the probes which are derived therefrom, at a level significantly greater than the background noise. The background noise may be associated with the hybridization of other polynucleotides present, for example other cDNAs present in a cDNA library. The level of the signal generated by the interaction between the polynucleotide capable of hybridizing selectively and the polynucleotides defined by the sequences SEQ ID NOS: above according to the invention, or the probes, is generally 10 times, preferably 100 times, more intense than that generated by the interaction with other DNA sequences generating the background noise. The level of interaction can be measured, for example, by labeling the polynucleotides described above or the probes with radioactive elements, such as ³²P. Selective hybridization is generally obtained using very severe conditions for the medium (for example 0.03 M NaCl and 0.03 M sodium citrate at approximately 50° C.-60° C.).

The invention also comprises isolated polynucleotides encoding arogenate dehydrogenase enzymes, and homologs of the polynucleotides described above. According to the invention, the term “homolog” is intended to mean polynucleotides exhibiting one or more sequence modifications compared to the nucleotide sequences described above and encoding an enzyme with functional arogenate dehydrogenase activity. These modifications may be natural or obtained artificially according to the usual techniques of mutation leading in particular to the addition, deletion or substitution of one or more nucleotides compared to the sequences of the invention. These modifications determine a degree of homology with respect to the sequences described above. Advantageously, the degree of homology will be at least 70% compared to the sequences described, preferably at least 80%, more preferentially at least 90%. The methods for measuring and identifying homologies between nucleic acid sequences are well known to those skilled in the art. Use may, for example, be made of the PILEUP or BLAST programs (Basic Local Alignment Search Tool; Altschul et al., 1993, J. Mol. Evol. 36: 290-300; Altschul et al., 1990, J. Mol. Biol. 215: 403-10; see also http://www.ncbi.nlm.nih.gov/BLAST/).

The present invention also relates to fragments of the polynucleotides described above. The term “fragment” denotes in particular a fragment of at least 20 nucleotides, in particular of at least 50 nucleotides, and preferably of at least 100 nucleotides.

According to a particular embodiment of the invention, the polynucleotide according to the invention is represented by the sequence identifier SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 12.

The present invention also relates to polynucleotides comprising at least one of the polynucleotides as described above.

All the polynucleotides described above encode arogenate dehydrogenase enzymes. Consequently, the invention therefore extends to all the arogenate dehydrogenase enzymes encoded by all of these polynucleotides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic of the arogenate pathway.

FIG. 2 shows graphs of the enzymatic activity of TyrA-ATc, TyrA-AT1 and TyrA-AT2.

FIG. 3 shows a table of enzymatic activity of TyrA-ATc, TyrA-AT1 and TyrA-AT2.

FIG. 4 shows a table of Synechocystis arogenate dehydrogenase enzymatic activity.

According to a particular embodiment of the invention, the arogenate dehydrogenase enzyme is an enzyme the peptide sequence of which is selected from the sequence described by the sequence identifier SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13., or a fragment of these sequences. The term “fragment” is intended to mean essentially a biologically active fragment, i.e. a fragment of the sequence of an arogenate dehydrogenase enzyme having the same activity as a complete arogenate dehydrogenase enzyme.

According to a particular embodiment of the invention, the polynucleotides and the arogenate dehydrogenase enzymes described above originate from plants. More particularly, they originate from plants of the Arabidopsis genus, preferably of the A. thaliana genus, or from plants of the Picea genus, preferably Picea glauca.

According to another particular embodiment of the invention, the polynucleotides and the arogenate dehydrogenase enzymes described above originate from bacteria. More particularly, they originate from bacteria of the Synechocystis genus.

The present invention also relates to a chimeric gene comprising, functionally linked to one another, at least one promoter which is functional in a host organism, a polynucleotide encoding an arogenate dehydrogenase enzyme as defined in the present invention, and a terminator element which is functional in this same host organism. The various elements that a chimeric gene may contain are, firstly, elements which regulate the transcription, translation and maturation of proteins, such as a promoter, a sequence encoding a signal peptide or a transit peptide, or a terminator element constituting a polyadenylation signal and, secondly, a polynucleotide encoding a protein. The expression “functionally linked to one another” means that said elements of the chimeric gene are linked to one another in such a way that the functioning of one of these elements is affected by that of another. By way of example, a promoter is functionally linked to a coding sequence when it is capable of affecting the expression of said coding sequence. The construction of the chimeric gene according to the invention and the assembly of its various elements can be carried out using techniques well known to those skilled in the art, in particular those described by Sambrook et al., (1989, Molecular Cloning: A Laboratory Manual, Nolan C. ed., New York: Cold Spring Harbor Laboratory Press). The choice of the regulatory elements constituting the chimeric gene depends essentially on the host species in which they must function, and those skilled in the art are capable of selecting regulatory elements which are functional in a given host organism. The term “functional” is intended to mean capable of functioning in a given host organism.

The promoters which the chimeric gene according to the invention can contain are either constitutive or inducible. A constitutive promoter according to the present invention is a promoter which induces the expression of a coding sequence in all the tissues of a host organism and continuously, i.e. throughout the duration of the life cycle of said organism. Some of these promoters may be tissue-specific, i.e. express the coding sequence continuously, but only in a particular tissue of the host organism. Constitutive promoters may originate from any type of organism. Among the constitutive promoters which can be used in the chimeric gene of the present invention, mention may, for example, be made of bacterial promoters, such as that of the octopine synthase gene or that of the nopaline synthase gene, of viral promoters, such as that of the gene controlling transcription of the 19S or 35S RNAs of the cauliflower mosaic virus (Odell et al., 1985, Nature, 313, 810-812), or the promoters of the cassava vein mosaic virus (as described in patent application WO 97/48819). Among the promoters of plant origin, mention will be made of the promoter of the ribulose-biscarboxylase/oxygenase (RuBisCO) small sub-unit gene, the promoter of a histone gene as described in application EP 0 507 698, or the promoter of a rice actin gene (U.S. Pat. No. 5,641,876).

According to another particular embodiment of the invention, the chimeric gene contains an inducible promoter. An inducible promoter is a promoter which only functions, i.e. which only induces expression of a coding sequence, when it is itself induced by an inducing agent. This inducing agent is generally a substance which can be synthesized in the host organism subsequent to a stimulus external to said organism, this external stimulus possibly being, for example, a pathogenic agent. The inducing agent may also be a substance external to this host organism, capable of penetrating into this host organism. Advantageously, the promoter used in the present invention is inducible subsequent to an attack on the host organism by a pathogenic agent. Such promoters are known, such as, for example, the promoter of the plant O-methyl-transferase class II (COMT II) gene described in patent application FR 99 03700, the Arabidopsis PR-1 promoter (Lebel et al., 1998, Plant J. 16(2):223-233), the EAS4 promoter of the tobacco sesquiterpene synthase gene (Yin et al., 1997, Plant Physiol. 115(2): 437-451), or the promoter of the gene encoding 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (Nelson et al., 1994, Plant Mol. Biol. 25(3): 401-412).

Among the terminator elements which may be used in the chimeric gene of the present invention, mention may, for example, be made of the nos terminator element of the gene encoding Agrobacterium tumefaciens nopaline synthase (Beven et al., 1983, Nucleic Acids Res. 11(2), 369-385), or the terminator element of a histone gene as described in application EP 0 633 317.

It also appears to be important for the chimeric gene to additionally comprise a signal peptide or a transit peptide which makes it possible to control and orient the production of the arogenate dehydrogenase enzyme specifically in a part of the host organism, such as, for example, the cytoplasm, a particular compartment of the cytoplasm, or the cell membrane or, in the case of plants, in a particular type of cellular compartment, for example the chloroplasts, or in the extracellular matrix.

The transit peptides can be either single or double. The double transit peptides are optionally separated by an intermediate sequence, i.e. they comprise, in the direction of transcription, a sequence encoding a transit peptide of a plant gene encoding an enzyme located in plastids, a portion of sequence of the mature N-terminal portion of a plant gene encoding an enzyme located in plastids, and then a sequence encoding a second transit peptide of a plant gene encoding an enzyme located in plastids. Such double transit peptides are, for example, described in patent application EP 0 508 909.

Signal peptides of use according to the invention which may be mentioned include in particular the signal peptide of the tobacco PR-1α gene described by Cornelissen et al. (1987, Nucleic Acid Res. 15, 6799-6811), in particular when the chimeric gene according to the invention is introduced into plant cells or plants, or the signal peptide of the Mat α1 factor precursor (Brake et al., 1985, In: Gething M.-J. (eds.); Protein transport and secretion, pp. 103-108, Cold Spring Harbor Laboratory Press, New York), when the chimeric gene according to the invention is introduced into yeast.

The present invention also relates to a vector containing a chimeric gene according to the invention. The vector according to the invention is of use for transforming a host organism and expressing an arogenate dehydrogenase enzyme in this host organism. This vector may be a plasmid, a cosmid, a bacteriophage or a virus. In general, the main qualities of this vector should be an ability to persist and to self-replicate in the host organism's cells, in particular by virtue of the presence of an origin of replication, and to express therein an arogenate dehydrogenase enzyme. The choice of such a vector and also the techniques for inserting the chimeric gene according to the invention therein are widely described in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Nolan C. ed., New York: Cold Spring Harbor Laboratory Press) and are part of the general knowledge of those skilled in the art. Advantageously, the vector used in the present invention also contains, in addition to the chimeric gene of the invention, a gene encoding a selectable marker. This selectable marker makes it possible to select the host organisms effectively transformed, i.e. those having incorporated the vector. According to a particular embodiment of the invention, the host organism to be transformed is a microorganism, in particular a yeast, a bacterium, a fungus or a virus. According to another embodiment, the host organism is a plant or a plant cell. Among the genes encoding selectable markers which can be used, mention may be made of genes for resistance to antibiotics, such as, for example, the hygromycin phosphotransferase (Gritz et al., 1983, Gene 25: 179-188), but also the genes for tolerance to herbicides, such as the bar gene (White et al., NAR 18: 1062, 1990) for tolerance to bialaphos, the EPSPS gene (U.S. Pat. No. 5,188,642) for tolerance to glyphosate or else the HPPD gene (WO 96/38567) for tolerance to isoxazoles. Mention may also be made of genes encoding readily identifiable enzymes such as the GUS enzyme, or genes encoding pigments or enzymes which regulate the production of pigments in the transformed cells. Such selectable marker genes are in particular described in patent applications WO 91/02071, WO 95/06128, WO 96/38567 and WO 97/04103.

The present invention also relates to transformed host organisms containing a vector as described above. The term “host organism” is intended to mean any lower or higher monocellular or pluricellular organism into which the chimeric gene according to the invention can be introduced, so as to produce arogenate dehydrogenase enzyme. They are in particular bacteria, for example Escherichia coli, yeast, in particular of the Saccharomyces, Kluyveromyces or Pichia genera, fungi, in particular Aspergillus, a baculovirus, or preferably plant cells and plants.

According to the invention, the term “plant cell” is intended to mean any cell derived from a plant and able to constitute undifferentiated tissues such as calluses, differentiated tissues such as embryos, parts of plants, plants or seeds.

According to the invention, the term “plant” is intended to mean any differentiated multicellular organism capable of photosynthesis, in particular monocotyledons or dicotyledons.

The term “transformed host organism” is intended to mean a host organism which has incorporated into its genome the chimeric gene of the invention and consequently produces an arogenate dehydrogenase enzyme in its tissues, or in a culture medium. Those skilled in the art can use one of the many known methods of transformation to obtain the host organisms according to the invention.

One of these methods consists in bringing the cells to be transformed into contact with polyethylene glycol (PEG) and the vectors of the invention (Chang and Cohen, 1979, Mol. Gen. Genet. 168(1), 111-115); Mercenier and Chassy, 1988, Biochimie 70(4), 503-517). Electroporation is another method, which consists in subjecting the cells or tissues to be transformed and the vectors of the invention to an electric field (Andreason and Evans, 1988, Biotechniques 6(7), 650-660; Shigekawa and Dower, 1989, Aust. J. Biotechnol. 3(1), 56-62). Another method consists in directly injecting the vectors into the host cells or tissues by microinjection (Gordon and Ruddle, 1985, Gene 33(2), 121-136). Advantageously, the “biolistic” method may be used. In consists in bombarding cells or tissues with particles onto which the vectors of the invention are adsorbed (Bruce et al., 1989, Proc. Natl. Acad. Sci. USA 86(24), 9692-9697; Klein et al., 1992, Biotechnology 10(3), 286-291; U.S. Pat. No. 4,945,050). Preferentially, the plant transformation will be carried out using bacteria of the Agrobacterium genus, preferably by infecting the cells or tissue of said plants by A. tumefaciens (Knopf, 1979, Subcell. Biochem. 6, 143-173; Shaw et al., 1983, Gene 23(3): 315-330) or A. rhizogenes (Bevan and Chilton, 1982, Annu. Rev. Genet. 16: 357-384; Tepfer and Casse-Delbart, 1987, Microbiol. Sci. 4(1), 24-28). Preferentially, the transformation of plant cells with Agrobacterium tumefaciens is carried out according to the protocol described by Ishida et al. (1996, Nat. Biotechnol. 14(6), 745-750).

Those skilled in the art will choose the appropriate method as a function of the nature of the host organism to be transformed.

The present invention therefore also relates to a method for preparing the arogenate dehydrogenase enzyme, comprising the steps of culturing a transformed host organism comprising a gene encoding an arogenate dehydrogenase enzyme as defined above, in a suitable culture medium, recovering the arogenate dehydrogenase enzyme produced from the culture medium by centrifugation or by filtration, and then purifying the recovered enzyme by passing it through at least one chromatography column. These steps bring about the extraction and the purification, which may be total or partial, of the arogenate dehydrogenase enzyme obtained. Preferentially, the transformed organism is a microorganism, in particular a bacterium, a yeast, a fungus or a virus.

The present invention also comprises a method for identifying a herbicidal compound having as a target an arogenate dehydrogenase enzyme, characterized in that:

-   -   (a) at least two samples, each containing an equivalent amount         of arogenate dehydrogenase enzymes in solution, are prepared;     -   (b) one of the samples is treated with a compound;     -   (c) the arogenate dehydrogenase activity is measured in each one         of said samples;     -   (d) the compound used in step (b) is identified as being a         herbicidal compound when the activity measured in step (c) is         significantly less in the treated sample compared to the         untreated sample;     -   (e) the herbicidal activity of the compound identified in         step (d) is validated by treating plants with said compound.

According to the present method, the measurement of the arogenate dehydrogenase activity is carried out by any method which makes it possible either to measure a decrease in the amount of arogenate substrate, or to measure an accumulation of a product derived from the enzyme reaction, namely L-tyrosine or the cofactor NADPH. In particular, the measurement of the arogenate dehydrogenase activity can be carried out by the method described in example 4. In addition, the herbicidal activity validated in step (e) of the present method may be a lethal activity resulting in the death of the treated plant, or an activity which significantly slows down the growth of the treated plant.

According to the invention, the term “compound” is intended to mean any chemical compound or mixture of chemical compounds, including peptides and proteins. According to the invention, the term “mixture of compounds” is understood to mean at least two different compounds, such as, for example, the (dia)stereoisomers of a molecule, mixtures of natural origin derived from the extraction of biological material (plants, plant tissues, bacterial cultures, yeast cultures or fungal cultures, insects, animal tissues, etc.) or unpurified or totally or partially purified reaction mixtures, or else mixtures of products derived from combinatorial chemistry techniques.

According to a particular embodiment of the method according to the invention, the arogenate dehydrogenase enzymes used originate from plants, preferably from Arabidopsis thaliana.

According to another embodiment of the method according to the invention, the arogenate dehydrogenase enzymes used originate from bacteria, preferably bacteria of the Synechocystis genus.

Preferably, the arogenate dehydrogenase enzymes used in the method according to the invention are the enzymes according to the present invention, in particular those represented by SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13.

The invention also extends to the herbicidal compounds identified using the method mentioned above, in particular the herbicidal compounds having as a target an arogenate dehydrogenase enzyme, i.e. those which inhibit the activity of this enzyme. Preferentially, the herbicidal compounds are not general enzyme inhibitors. Also preferentially, the herbicidal compounds according to the invention are not compounds already known to have herbicidal activity.

The present invention also relates to herbicidal agrochemical compositions comprising, as active material, at least an effective amount of a herbicidal compound according to the invention.

According to the invention, the term “herbicidal agrochemical composition” is intended to mean a composition which can be applied preventatively or curatively to the areas on which cultivated plants are being or must be grown, in order to prevent the development of undesirable plants or “weeds” on the areas on which said cultivated plants are grown, whatever their state of development. An effective amount of herbicidal compound according to the invention corresponds to an amount of compound which makes it possible to destroy or inhibit the growth of the undesirable plants.

The herbicidal agrochemical compositions according to the invention comprise a herbicidal compound according to the invention or one of its agriculturally acceptable salts or a metal or metalloid complex of this compound, in combination with an agriculturally acceptable solid or liquid carrier and/or a surfactant, also agriculturally acceptable. In particular, the usual inert carriers and the usual surfactants can be used. These compositions cover not only the compositions ready to be applied to a plant or a seed to be treated using a suitable device, such as a spraying or dusting device, but also the concentrated commercially available compositions which must be diluted before they are applied to the crop.

The herbicidal compositions according to the invention may also contain many other ingredients, such as, for example, protective colloids, adhesives, thickeners, thixotropic agents, penetrating agents, stabilizers or sequestering agents. More generally, the active materials can be combined with any solid or liquid additives which comply with the usual formulating techniques.

According to the present invention, the term “carrier” denotes a natural or synthetic, organic or inorganic material with which the active material is combined in order to facilitate its application to the parts of the plant. This carrier is therefore generally inert and it must be agriculturally acceptable. The carrier may be solid (for example clays, natural or synthetic silicates, silica, resins, waxes, solid fertilizers) or liquid (for example water, alcohols, in particular butanol).

The surfactant may be an emulsifier, dispersing agent or wetting agent of the ionic or nonionic type, or a mixture of such surfactants. Mention may, for example, be made of polyacrylic acid salts, lignosulfonic acid salts, phenolsulfonic or naphthalenesulfonic acid salts, polycondensates of ethylene oxide with fatty alcohols or with fatty acids or with fatty amines, substituted phenols (in particular alkylphenols or arylphenols), salts of sulfosuccinic acid esters, taurine derivatives (in particular alkyl taurates), phosphoric esters of alcohols or of phenols which are polyoxyethylated, esters of fatty acids and of polyols, and derivatives of the above compounds containing sulfate, sulfonate and phosphate functions. The presence of at least one surfactant is generally essential when the active material and/or the inert carrier are not water-soluble and when the vector agent for the application is water.

The present invention also relates to transgenic plants tolerant to a herbicidal compound having as a target an enzyme involved in one of the metabolic steps of conversion of prephenate to L-tyrosine, characterized in that they contain a gene encoding a prephenate dehydrogenase enzyme and express said enzyme in their tissue. A prephenate dehydrogenase enzyme is an enzyme which catalyzes the reaction of conversion of prephenate to p-hydroxyphenylpyruvate. The identification of an enzyme with prephenate dehydrogenase activity can be carried out by any method which makes it possible either to measure a decrease in the amount of the prephenate substrate, or to measure an accumulation of a product derived from the enzyme reaction, namely p-hydroxyphenylpyruvate or one of the cofactors NADH or NADPH. In particular, the measurement of the prephenate dehydrogenase activity can be carried out using the method described in example 4.

According to a particular embodiment of the invention, the transgenic plants according to the invention are tolerant with respect to a herbicidal compound having as a target an arogenate dehydrogenase enzyme, preferably an arogenate dehydrogenase enzyme as described in the present invention.

According to another particular embodiment of the invention, the transgenic plants according to the invention are tolerant with respect to a herbicidal compound having as a target a prephenate aminotransferase enzyme.

According to a particular embodiment of the invention, the gene encoding the prephenate dehydrogenase enzyme expressed in the tolerant plants according to the invention is a yeast gene. Preferably, it is the gene encoding the Saccharomyces cerevisiae prephenate dehydrogenase enzyme (accession No. NC001134) as described in Mannhaupt et al. (1989, Gene 85, 303-311) and represented by the sequence identifier SEQ ID NO: 14.

According to another particular embodiment of the invention, the gene encoding the prephenate dehydrogenase enzyme expressed in the tolerant plants according to the invention is a bacterial gene. Preferably, it is a gene from a bacterium of the Bacillus genus, in particular of the species B. subtilis (accession No. M80245) as represented by the sequence identifier SEQ ID NO: 16. Preferably, it is a gene from a bacterium of the Escherichia genus, in particular of the species E. coli (accession No. M10431) as described in Hudson et al. (1984, J. Mol. Biol. 180(4), 1023-1051) and represented by the sequence identifier SEQ ID NO: 18. Preferably, it is a gene from a bacterium of the Erwinia genus, in particular the species E. herbicola (accession No. 43343) as represented by the sequence identifier SEQ ID NO: 20.

According to particular embodiment of the invention, the gene encoding the prephenate dehydrogenase enzyme expressed in the tolerant plants according to the invention is a fungal gene.

The transgenic plants according to the invention are obtained by genetic transformation with a gene encoding a prephenate dehydrogenase enzyme. Preferably, this gene is a chimeric gene comprising, functionally linked to one another, at least one promoter which is functional in a host organism, a polynucleotide encoding a prephenate dehydrogenase enzyme, and a terminator element which is functional in this same host organism. This gene is generally introduced into a vector, which is used to introduce said gene into said plants by one of the methods of transformation described above.

The present invention also relates to a method for producing plants tolerant with respect to herbicidal compounds having as a target an enzyme involved in one of the metabolic steps for conversion of prephenate to L-tyrosine, characterized in that said plants are transformed with a gene encoding a prephenate dehydrogenase enzyme in such a way that they express it in their tissues.

According to a particular embodiment of the invention, the present method applies to the production of plants tolerant with respect to a herbicidal compound having as a target an arogenate dehydrogenase enzyme as described in the present invention.

According to another particular embodiment of the invention, the present method applies to the production of plants tolerant with respect to a herbicidal compound having as a target a prephenate aminotransferase enzyme.

The present method therefore also comprises a method for producing plants tolerant with respect to a herbicidal compound having as a target an arogenate dehydrogenase enzyme, characterized in that said plants are transformed with a gene encoding a prephenate dehydrogenase enzyme in such a way that they express it in their tissues.

The transgenic plants according to the invention may also contain, in addition to a gene encoding a prephenate dehydrogenase enzyme, at least one other gene containing a polynucleotide encoding a protein of interest. Among these polynucleotides encoding a protein of interest, mention may be made of polynucleotides encoding an enzyme for resistance to a herbicide, for example the polynucleotide encoding the bar enzyme (White et al., NAR 18:1062, 1990) for tolerance to bialaphos, the polynucleotide encoding the EPSPS enzyme (U.S. Pat. No. 5,188,642; WO 97/04103) for tolerance to glyphosate, or else the polynucleotide encoding the HPPD enzyme (WO 96/38567) for tolerance to isoxazoles. Mention may also be made of a polynucleotide encoding an insecticidal toxin, for example a polynucleotide encoding a toxin of bacterium Bacillus thuringiensis (for example, see International Patent Application WO 98/40490). Other polynucleotides for resistance to diseases may also be contained in these plants, for example a polynucleotide encoding the oxaylate oxidase enzyme as described in patent application EP 0 531 498 or U.S. Pat. No. 5,866,778, or a polynucleotide encoding another antibacterial and/or antifungal peptide, such as those described in patent applications WO 97/30082, WO 99/24594, WO 99/02717, WO 99/53053 and WO 99/91089. Mention may also be made of polynucleotides encoding agronomic characteristics of the plant, in particular a polynucleotide encoding a delta-6 desaturase enzyme, as described in U.S. Pat. Nos. 5,552,306 and 5,614,313, and patent applications WO 98/46763 and WO 98/46764, or a polynucleotide encoding a serine acetyltransferase (SAT) enzyme, as described in patent applications WO 00/01833 and PCT/FR 99/03179.

The following examples make it possible to illustrate the present invention without, however, limiting the scope thereof.

EXAMPLE 1 Identification of the Gene Encoding the Arabidopsis Thaliana Arogenate Dehydrogense Enzyme

A comparison of the sequences of all the prephenate dehydrogenase and arogenate dehydrogenase enzymes currently available in the public databases revealed four short portions of homologous sequences. The enzymes compared are yeast prephenate dehydrogenase (accession number: Z36065), Bacillus subtilis prephenate dehydrogenase (accession number: M80245) and Synechocystis prephenate dehydrogenase (accession number: D90910). These portions of homology made it possible to identify an A. thaliana gene (accession number: AF096371) initially noted as encoding an enzyme “similar to the specific D-isomer 2-hydroxy acid dehydrogenase”. This gene consists of two exons separated by a 94 bp intron. The first exon comprises a 1.08 kb open reading frame containing a putative chioroplast transit peptide sequence located downstream of the first ATG codon. The second exon potentially encodes an 892 bp open reading frame. A very strong homology of approximately 60% exists between the protein sequences deduced from the two exons. This homology extends to 70% if the putative chloroplast transit peptide sequence located in the first exon is not taken into account. In addition, each one of the two predicted protein sequences has the size and possesses the four homologous portions characteristic of the prephenate/arogenate dehydrogenase enzymes. This gene was named TyrA (SEQ ID NO: 1).

EXAMPLE 2 Transcriptional Characterization of TyrA

The size of the transcript of the TyrA gene was determined using the Northern blotting and PCR techniques. Purified mRNAs extracted from young leaves of A. thialiana were hybridized with ³²P-radiolabeled probes corresponding to fragments of DNA of the two exons of TyrA. This analysis made it possible to identify a 1.8-1.9 kb transcript very close to the presumed size of an mRNA containing the two exons. In addition, although the complete cDNA could not be amplified by PCR, a 1.5 kb PCR fragment was obtained. This fragment comprises the 5′ oligonucleotide (P8 =5′-GCTAAAACTCTTCTCCTTCAATACTTACCTG-3′: SEO ID NO: 30) beginning at position 513 bp, and the 3′oligonucleotide (P7 =5′-CAGTATAATTAGTA-GTCAAGGATCCTGACTGAGAG-3′; SEQ ID NO: 29) complementary to the 3′UTR and beginning at position 2053 bp. This fragment contains a portion of the first coding sequence (TyrA-A T1) and the complete sequence of the second coding sequence (TyrA-AT2). Analysis of the sequence of this cDNA confirmed the splicing of the intron. The results of the analyses by Northern blotting and PCR strongly suggests the existence of an mRNA transcript containing the two coding regions TyrA -A T1 (SEQ ID NO: 4) and TyrA-AT2 (SEQ ID NO: 6).

EXAMPLE 3 Preparation of Constructs Containing the Various Coding Sequences of the A. Thaliana Arogenate Dehydrogenase

The first exon TyrA-AT1 was obtained by PCR amplification of the genomic DNA of A. thaliana with the oligonucleotide P1 (5′-TCTCCATATGATCTTTCAATCTCATTCTCATC-3′; SEQ ID NO: 32) which introduces an Nde I restriction site (underlined) at the first ATG codon, and the oligonucleotide P2 (5′-CTAACTAACTAACTACATACCTCATCATATCC-3′: SEQ ID NO: 24) which is complementary to the 3′ end of the first exon and to the 5′ end of the intron and introduces a stop codon (underlined). Three constructs lacking the sequence encoding the transit peptide were also produced with the oligonucleotide P3 (5′-CCTCTCTTTCCATATGCTCC-CTTCTC-3′SEQ ID NO: 25) which introduces an Nde I restriction site (underlined) at the second ATG codon (M43) at position 127, the oligonucleotide P4 (5′-CCGCCAGCCACCT-CCATATGACCGACACCATCC-3′; SEQ ID NO: 26) which introduces an ATG initiating codon and an Nde I restriction site (underlined) at position 174 from the first ATG codon (V58M), and the oligonucleotide P5 (5′-CGCCACCCCTCATATGCGTATCGCC-3′; SEQ ID NO: 27) which introduces an ATG initiating codon and an Nde I restriction site (underlined) at position 222 from the first ATG codon (L75M). All the OCR fragments corresponding to the first exon, which may or may not encode a transit peptide, were cloned into the plasmid pPCR-Script (Stratagene). Nde I-BamH I DNA fragments containing the coding sequences, with or without the transit peptide sequence, were then cloned into the plasmid pET21 a(+) (Novagen), leading to the development of the plasmids pET21-TyrA-AT1, with and without transit peptide sequence (pET21-TyrA-AT1-M1,pET21-TyrA-AT1-M43, pET21-TyrA-AT1-M58 and pET21-TyrA-ATi-M75).

Two other oligonucleotides were used to amplify the second coding sequence (TyrA-AT2). The oligonucleotide P6 (5′-GATGCATCTTTGCATATGATGAGGTCAGAAGATG-3′; SEQ ID NO: 28) introduces an Nde I restriction site (underlined) at the ATG codon of the second open reading frame (at position 1081 from the first ATG codon), and the oligo-nucleotide P7 (5′-CAGTATAATTAGTAGTCAAGGATCCTGACTGAGAG-3′; SEQ ID NO: 29), complementary to the start of the 3′-UTR, introduces a BamH I restriction site (underlined). The PCR fragment corresponding to the second coding sequence was digested with Nde I-BamH I and then cloned into the plasmid pET21 a(+), giving the plasmid pET21-TyrA-AT2.

The complete coding sequence was reconstituted by assembly of the missing 5′end of the first exon with a partial TyrA-AT cDNA (1.5 kb), obtained by PCR amplification of the Arabidopsis cDNA with the oligo-nucleotide P8 (5′-GCTAAAACTCTTCTCCTTCAATACTTACCTG-3′; SEQ ID NO: 30) beginning at position 513 bp from the first ATG codon, and the 3′oligonucleotide P7. An EcoRV restriction site located at position 812 bp from the first ATG codon and present in the 5′ end of the partial TyrA-ATc DNA was used for the reconstitution. The partial TyrA-AT cDNA was cloned into the plasmid pPCR-Script. An EcoRV-EcoRV fragment was obtained from the plasmid pPCR-Script-TyrA-AT and then cloned into the plasmid pPCR-Script-TyrA-AT1 digested beforehand with EcoRV. This manipulation led to the plasmid pPCR-Script-TyrA-ATc being obtained. An Ndel-BamHl fragment containing the complete coding sequence was excised from the plasmid pPCR-Script-TyrA-ATc, and then cloned into a plasmid pET21a(+) (Novagen), digested beforehand with Nde 1 and BamH 1, producing the plasmid pET21a(+)-TyrA-ATc. Then, in the same way as for the first exon, four plasmids pET21a(+)-TyrA-ATc were obtained; a plasmid containing the complete coding sequence with the sequence encoding the putative transit peptide, and three plasmids lacking this transit peptide sequence, which was cleaved at three different sites (M43, V58 and L75, see above).

For all the constructs described above, the cDNA inserts were sequenced in order to be sure that no unwanted mutation had been introduced during the PCR amplification.

EXAMPLE 4 Measurement of the Enzyme Activities

The arogenate dehydrogenase activity is measured at 25° C. by spectrophotometric monitoring, at 340 nm, of the formation of NADH or NADPH in a solution containing 50 mM of Tris-HCl, pH 8.6, 300 μm of arogenate and 1 mM of NAD or NADPH in a total volume of 200 μl.

The prephenate dehydrogenase activity is measured at 25° C. by spectrophotometric monitoring, at 340 nm, of the formation of NADH or NADPH in a solution containing 50 mM of Tris-HC1, pH 8.6, 300 μM of prephenate and 1 mM of NAD or NADPH in a total volume of 200 μl.

EXAMPLE 5 Production of Recombinant Arogenate Dehydrogenase

Eshcerichia coli AT2471 cells were transformed with each one of the plasmids pET21-TyrA-AT obtained in example 3, and then cultured at 37° C. in 2 liters of Luria-Bertani medium supplemented with 100 μg/ml of carbenicillin. When the culture had reached the equivalent of an absorbance at 600 nm (A600) of 0.6, 1 mM of isopropyl-β-D-thiogalactoside was added to the culture medium in order to induce recombinant protein synthesis. The cells were then cultured for 16 h at 28° C., harvested, and then centrifuged for 20 min at 40 000 g. The pellet was then resuspended in a 50 mM Tris-HC1 buffer, pH 7.5, containing 1 mM EDTA, 1 mM dithiothreitol, 1 mM benzamidine HCl and 5 mM aminocaproic acid, and then sonicated (100 pulses every 3 seconds at power 5) with a Vibra-Cell disrupter (Sonics and Materials, Danbury, Conn., USA). The crude extracts thus obtained were then centrifuged for 20 min at 40 000 g, and the supernatants were used directly for the enzyme assays.

The SDS-PAGE analyses of total protein extracts of the E. coli strain AT 2471 containing the various constructs pET21-TyrA-Atc, pET21-TyrA-AT1 and pET21-TyrA-AT2 revealed the presence of three recombinant proteins having molecular masses of 66-68 kDa, 35 kDa and 33-34 kDa, respectively. These molecular masses correspond well to the masses deduced from their respective coding sequences (68786 Da for TyrA-ATc, 34966 Da for Tyr-A-AT1, and 34069 Da for Tyr-A-AT2). For the transformants containing the complete coding sequence (TyrA-Atc) and the first coding sequence (TyrA-AT1), recombinant proteins were observed only with the constructs encoding the proteins M58-TyrA-Atc and M58-TyrA-AT1. The three recombinant proteins were mainly found in the protein bodies. However, the presence of small amounts of recombinant proteins in the soluble protein extracts of E. coli made it possible to characterize the biochemical properties.

EXAMPLE 6 Identification and Biochemical Characterization of the Arabidopsis Thaliana Arogenate Dehydrogenase Enzymes

The biochemical characterization of the recombinant arogenate dehydrogenase enzymes was carried out using the soluble protein extracts of the transformed E. coli strains. The arogenate dehydrogenase activity was measured according to the method described in example 4. A strictly NADP-dependent arogenate dehydrogenase activity was demonstrated for each one of the three recombinant enzymes. No arogenate dehydrogenase activity was detected in the presence of NAD, and no prephenate dehydrogenase activity was detected whatever the cofactor used (NADP or NAD) and whatever the protein tested (TyrA-ATc, TyrA-AT1 or TyrA-AT2). In addition, prephenate at a concentration of 1 mM does not inhibit the arogenate dehydrogenase activity of the three recombinant enzymes. Each one of these enzymes has a Michaelis-Menten-type behavior, and their Km value for arogenate and NADP is relatively the same (FIGS. 2 and 3). The Michaelis constants for NADP are, respectively, 40 μM for TyrA-Atc, 60 μM for TyrA-AT1, and 20 μM for TyrA-AT2. The Michaelis constants for arogenate are, respectively, 70 μM for TyrA-Atc, 45 μM for TyrA-AT1, and 45 μM for TyrA-AT2. In addition, like the other plant arogenate dehydrogenases (Byng et al., 1981, Phytochemistry 6, 1289-1292; Connelly and Conn, 1986, Z. Naturforsch 41c, 69-78; Gaines et al., 1982 Planta 156, 233-240), the Arabidopsis arogenate dehydrogenases are all very sensitive to tyrosine, the product of the enzyme reaction, and insensitive to 1 mM of phenylalanine and 1 mM of p-hydroxyphenylpyruvate. The inhibition by tyrosine is competitive with respect to arogenate (Ki of 14 μM for TyrA-Atc, 8 μM for TyrA-AT1, and 12 μM for TyrA-AT2), and noncompetitive with respect to NADP.

EXAMPLE 7 Identification and Biochemical Characterization of the Syfnechocystis Arogenate Dehydrogenase Enzyme

The sequence of the gene encoding the A. thaliana arogenate dehyrogenase identified in example 1 (TyrA) made it possible to identify an arogenate dehydrogenase gene in the bacterium Synechocystis (accession number: 1652956). This gene was originally described as encoding a “prephenate dehydrogenase” enzyme. It was isolated from a Synechocystis genomic library and the enzyme was produced in the same way as the A. thaliana enzyme, according to the protocol described in example 5 . Biochemical characterization of the enzyme produced made it possible to demonstrate that it is an arogenate dehydrogenase enzyme and not a prephenate dehydrogenase enzyme. This biochemical characterization of the Synechocystis arogenate dehydrogenase enzyme was carried out using the purified soluble protein extracts of the transformed E. coli strains. The arogenate dehydrogenase activity was measured according to the method described in example 4 . A strictly NADP-dependent arogenate dehydrogenase activity was demonstrated for this enzyme. No arogenate dehydrogenase activity was detected in the presence of NAD, and no prephenate dehydrogenase activity was detected whatever the cofactor used (NADP or NAD). In addition, prephenate at a concentration of 1 mM does not inhibit the arogenate dehydrogenase activity of this enzyme. The Synechocystis arogenate dehydrogenase has a Michaelis-Menten-type behavior (FIG. 4). The Michaelis constant is 6 μM for NADP, and 107 μM for arogenate.

EXAMPLE 8 Identification of Other Plant Arogenate Dehydrogenase Enzymes

The sequence of the gene encoding the A. thaliana arogenate dehydrogenase identified in example 1 (TyrA) made it possible to identify another arogenate dehydrogenase gene in A. thaliana. This new gene (accession number: AC0342561; SEQ ID NO: 8) was initially noted as “containing similarity with the embryo abundance protein (EMB20) of Picea glauca”. It also has a putative chloroplast transit peptide sequence, but no repeat region.

The sequence of the TyrA gene also made it possible to identify two other cDNAs encoding arogenate dehydrogenase enzymes in the public EST (Expressed Sequence Tags) databases. One of these cDNAs, which is not complete, corresponds to a tomato cDNA (TC41067; SEQ ID NO: 22). The incomplete nature of this cDNA does not make it possible to determine whether it is duplicated like TyrA, since its 3′ end stops just after the codon corresponding to D356 of Tyr-AT1. The second cDNA corresponds to a complete cDNA of Picea glauca (accession number: L47749; SEQ ID NO: 10) and does not possess a repeat region. This Picea glauca cDNA was noted as being an “embryo abundance protein”. 

1. A method for identifying a herbicidal compound having as a target an enzyme with arogenate dehydrogenase activity, said method comprising the steps of (a) preparing at least two samples, each containing an equivalent amount of an arogenate dehydrogenase enzyme in solution; (b) treating one of the samples with a compound; (c) measuring the arogenate dehydrogenase activity in each one of said samples; (d) comparing the activity of the treated sample with the activity of the untreated sample or samples, wherein the compound used in step (b) is identified as being a herbicidal compound when the activity measured in step (c) is significantly less in the treated sample compared to the untreated sample or samples; and (e) treating plants with the compound identified in step (d) to validate the herbicidal activity of said compound.
 2. The method as claimed in claim 1, wherein the arogenate dehydrogenase enzyme in step (a) is is encoded by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO:
 8. 